ABSTRACT Craniomaxillofacial (CMF) defects present unique, unmet challenges to the field of tissue engineering. Typically large in size and characterized by significant loss of hard- and soft-tissue, severe CMF defects are prevalent after acute (tumor resection, trauma) and chronic (degenerative, infectious) injuries. We are developing a collagen-based biomaterial to increase the quality and speed of bone regeneration as the essential first step for creating advanced biomaterials for complex CMF defects. Autologous bone transplantation remains the current gold-standard to repair structural CMF defects. However, limited access to autologous bone, concerns with secondary wound site creation, the destructive impact of post-injury inflammatory processes, and the irregular geometry of CMF defects motivate our efforts. Our long-term goal is to demonstrate a biomaterial that actively instructs, rather than passively supports, osteogenic differentiation and new bone formation using a patient's own mesenchymal stem cells (MSCs). However, while MSCs show promise for aiding healing, inflammatory signals within the wound can significantly reduce their efficacy. Our immediate objective is to demonstrate the innovative use of allogeneic tissue sources to create regeneration- inducing biomaterials that address two critical hurdles: [1] promoting MSC-osteogenesis; and [2] altering the kinetics of the M1-to-M2 macrophage transition through biomaterial design to further improve regeneration. We have recently developed a mineralized collagen scaffold with significant potential for regenerative repair of bone that can promote MSC osteogenesis in the absence of traditional growth factor supplements. Here we will integrate matrix proteins derived from the amniotic membrane (AM), the innermost layer of the placenta known to have significant immunomodulatory and anti-scarring properties, with this mineralized collagen scaffold to create a bioactive composite. We hypothesize the mineralized CG-AM composite will enhance MSC osteogenesis via endogenous BMPR activation and promote M2-like macrophage phenotype in response to inflammatory challenge. In Aim 1 we will dissect the contribution of AM matrix on osteogenic differentiation and inflammatory response within a mineralized collagen scaffold. In Aim 2 we will define the quality and kinetics of CG-AM composite induced mandible bone regeneration. While decellularized AM has shown promise as a stand-alone product for soft tissue repair, its significance as a bioactive component in 3D biomaterials has not been extensively investigated. We employ a systematic means to incorporate AM-matrix into the CG biomaterial to create a mineralized CG-AM composite, and then will define the contribution of the composite on MSC osteogenic differentiation, macrophage activity, and mandible regeneration in a clinically relevant porcine mandible defect model. Results derived here will significantly aid our ongoing efforts to design shelf-stable biomaterials that can be used clinically to regenerate large craniomaxillofacial defects.